Microbial transformation or bioconversion reactions have long been used to facilitate the chemical synthesis of a wide variety of pharmaceutical products. Stereospecific reactions carried out under mild enzymatic conditions frequently offer advantages over comparable chemical processes which result in undesireable side products. Microorganisms also have the ability to carry out simultaneous independent or sequential reactions on a substrate molecule, minimizing the number of distinct steps in a synthesis and reducing the total cost of the desired intermediate or end product.
General features of microbial systems used as biocatalysts for the transformation of organic compounds has been reviewed (See e.g., Goodhue, Charles T., Microb. Transform. Bioact. Compd., 1:9-44, 1982). Biotransformations can be carried out, for example, in continuous cultures or in batch cultures. Enzymes secreted from the microorganism react with a substrate, and the product can be recovered from the medium. Intracellular enzymes can also react with a substrate if it is able to enter cells by an active or a passive diffusion process. Immobilized, dried, permeabilized, and resting cells, and spores have also been used for microbial transformations. The use of cell extracts and purified enzymes in solution, or immobilized on carriers, may eventually offer significant cost or control advantages over traditional fermentation methods.
Bioconversion reactions have been widely used in the field of steroids (Kieslich, K.; Sebek, O. K. Annu. Rep. Ferment. Processes 3:275-304, 1979; Kieslich, Klaus. Econ. Microbiol., 5 (Microb. Enzymes Bioconvers.): 369-465, 1980). A variety of reactions have been characterized, including hydroxylation, epoxidation, oxidation, dehydrogenation, ring and side chain degradation, reduction, hydrolysis, and isomerization reactions. Many types of microorganisms have also been used including species as diverse, for example, as Acremonium, Aspergillus, Rhizopus, Fusarium, Penicillium, Streptomyces, Actinomyces, Nocardia, Pseudomonas, Mycobacterium, Arthrobacter and Bacillus. 
A variety of approaches have been used to facilitate the hydroxylation of intermediates used in the synthesis of commercially-important steroid compounds. U.S. Pat. No. 4,588,683, for example, describes a method of preparing 11 beta, 17 alpha, 20, 21 tetrahydroxy steroids by incubating substrate compounds in a medium comprising a fungal culture of the genus Curvularia capable of effecting 11 beta hydroxylation. Aspergillus ochraceus cultures and preparations of mycelia have also been used to convert progesterone and other steroids to their corresponding 11 alpha hydroxy forms (Tan, L. and Falardeau, P., 1970; Tan L., and Falardeau P., J. Steroid Biochem. 1:221-227, 1970; Samanta, T. B. et al., Biochem. J. 176, 593-594, 1978; Jayanthi, C. R. et al., Biochem. Biophys. Res. Commun. 106:1262-1268, 1982).
The advent of new and expanded clinical uses of steroids for the treatment of a wide variety of disorders has created a need for improved methods for the production of steroid compounds and their intermediates on a commercial scale. U.S. Pat. No. 4,559,332, for example, describes a number of methods for the preparation of 20-spiroxane series of steroid compounds, including methods for the preparation of eplerenone methyl hydrogen 9,11α-epoxy-17α-hydroxy-3-oxopregn-4-ene-7α,21-dicarboxylate, γ-lactone (also referred to as eplerenone or epoxymexrenone) and related compounds. WO 98/25948 and U.S. application Ser. No. 09/319,673 describe novel processes for the preparation of 9,11-epoxy steroid compounds, especially those of the 20-spiroxane series and their analogs, novel intermediates useful in the preparation of steroid compounds, and processes for the preparation of such novel intermediates. U.S. Pat. No. 6,046,023 discloses improved methods for the microbial transformation of canrenone or estr-4-ene-3,17-dione into its 11 α-hydroxy analogue using microorganisms of the genus Aspergillus, Rhizopus, and Pestelotia, using steroid substrates having a purity of less than 97% and more than 90% at a concentration greater than 10 g/L.
Many modern, systematic approaches needed to optimize bioconversion of particular steroid intermediates are often hindered by insufficient biochemical knowledge of the enzymes involved in their synthesis and degradation. Eukaryotic cytochromes P450 appear to be associated with the endoplasmic reticulum (ER) or mitochondrial membranes. The electron donor for ER-associated cytochrome P450 enzymes is often an FAD/FMN-dependent NADPH-cytochrome P450 oxidoreductase. Electron transfer in the mitochondrial cytochromes P450 is usually mediated by an NADPH-ferredoxin oxidoreductase and ferrodoxin. The specific electron donors known to be involved in mammalian steroidogenesis, are also called adrenodoxin reductase and adrenodoxin, respectively.
While fungal biotransformations are known to be mediated by cytochrome P450 enzymes, many of these enzymes are extremely difficult to purify in an enzymatically-active form (van den Brink et al., Fungal Genetics and Biology 23, 1-17, 1998). Many fungal P450 enzymes appear to be associated with the endoplasmic reticulum (van den Brink et al., Fungal Genetics and Biology 23, 1-17, 1998). Yeast have an adrenodoxin reductase homologue which was shown to couple with a mammalian 11 beta hydroxylase in vitro. (Lacour et al., Journal of Biological Chemistry 273, 23984-23992, 1998). In contrast, the electron donor which couples with Aspergillus ochraceus 11 alpha hydroxylase was predicted to be an NADPH-cytochrome P450 oxidoreductase (Samanta and Ghosh, J Steroid Biochem 28, 327-32, 1987). The steroid 11 alpha hydroxylation complex in Rhizopus nigricans also appears to require an NADPH-cytochrome p450 oxidoreductase (Makovec and Breskvar, Arch Biochem Biophys. 357, 310-6, 1998). Amplification of cytochrome R. nigricans P450 and NADPH-cytochrome P450 reductase activities in preparations of progesterone-induced fungal mycelia may the facilitate biochemical characterization of both enzymes (Makovec and Breskvar, Pflugers Arch—Eur J. Physiol 439(Suppl): R111-R112, 2000).
Aspergillus ochraceus spores have been shown to catalyze the 11 alpha hydroxylation of steroid substrates such as progesterone (Dutta T K, Datta J, Samanta T B, Biochem. Biophys. Res. Commun. 192:119-123, 1993). A. fumigatus is also known to exhibit a steroid 11 alpha hydroxylase activity (Smith et al., J Steroid Biochem Mol Biol 49:93-100, 1994). The A. fumigatus enzyme is distinguished from the A. ochraceus enzyme, in that it appears to be a cytochrome P450 with dual site-specificity for 11 alpha and 15 beta hydroxylation and, unlike the A. ochraceus hydroxylase, appears to be non-inducible.
Despite recent advances in sequencing technologies, detailed knowledge about the structural relationships of fungal cytochrome P450s gleaned from nucleotide sequence data remains primitive. Breskvar et al., (Biochem. Biophys. Res. Commun 1991; 178, 1078-1083, 1991) have described a genomic DNA sequence from Rhizopus nigricans for a putative P-450 encoding an 11α-hydroxylase for progesterone. This sequence may not be complete, however, since the predicted amino acid sequence lacks the canonical heme-binding motif, FxxGxxxCxG, which is common to almost all known cytochrome P-450 enzymes. (Nelson et al, Pharmacogenetics 6:1-42, 1996).
The cloning and characterization of the NADPH cytochrome P450 oxidoreductase (cprA) gene of Aspergillus niger has been described (van den Brink, J., et al., Genbank accession numbers Z26938, CAA81550, 1993, unpublished). The primary structure of Saccharomyces cereuisiae NADPH-cytochrome P450 reductase has also been deduced from the nucleotide sequence of its cloned gene (Yabusaki et al., J. Biochem. 103(6): 1004-1010, 1988).
Several other approaches have been used to facilitate the cloning and analysis of steroid enzymes. U.S. Pat. Nos. 5,422,262, 5,679,521, and European patent EP 0 528 906 B1, for example, describes the expression cloning of steroid 5 alpha reductase, type 2. U.S. Pat. No. 5,869,283, for example, describes an expression cassette comprising heterologous DNAs encoding two or more enzymes, each catalyzing an oxidation step involved conversion of cholesterol into hydrocortisone, including the conversion of cholesterol to pregnenolone; the conversion of pregnenolone to progesterone; the conversion of progesterone to 17 α-hydroxy-progesterone; the conversion of 17α-hydroxyprogesterone to cortexolone; and the conversion of cortexolone to hydrocortisone.
The sequences of Aspergillus ochraceus 11 alpha hydroxylase and A. ochraceus oxidoreductase have not been reported. Knowledge about their sequence could greatly facilitate the development of expression vectors and recombinant host strains that can carry out more efficient bioconversions of steroid intermediates and the synthesis of end products on a commercial scale without the problems associated with partially-characterized host strains or an incomplete understanding of the enzymes involved in steroidogenesis. The present invention overcomes many of the limitations discussed above by identifying enzymes capable of carrying out the 11 alpha hydroxylation of steroids. This approach not only greatly facilitates the use of 11 alpha hydroxylation, but also permits the development of new strategies for the identification of similar enzymes from other fungi, the cloning of other enzymes involved in steroidogenesis from Aspergillus ochraceus and other microorganisms, and the development of improved host strains or methods using free cells or immobilized cells or enzymes in bioconversion reactions. Similar approaches could also be developed to aid in the construction of expression vectors and recombinant host strains that are more amenable to propagation and control than wild-type microorganisms now commonly used for bioconversion in large scale bioreactors.